Experiments on the design and management of field trials of transgenic crops
Experiments on the design and management of field trials of transgenic crops
With the development of transgenic technology systems, the transfer of heterologous genes into the plant genome has become possible, which provides a new avenue for plant breeders to expand their genetic resources. The design and management of field trials of transgenic crops vary depending on the purpose. Breeders analyze the stability of genotypes and phenotypes of transgenic crops and record changes in the number of transgenic individuals under field conditions. The source of this experiment is "A Guide to Transgenic Technology and Field Identification Experiments in Wheat Crops" [English] H.D. Jones P.R . Hewley, eds.
Operation method
Design and management of field trials of transgenic crops
Materials and Instruments
transgenic strain Move 3.1 Geographic location requirements for release tests For more product details, please visit Aladdin Scientific website.
The release site should be a segregated area suitable for growing the crop, with flora and fauna that are not different from the surrounding farmland and without rare or protected plant or animal species, and it must also be ensured that there are no community habitats or protected areas recognized by a government agency in areas distant from the release site. The test area is required to be flat, so that the risk of gene escape can be reduced, but also so that it can be easily farmed, so that the test is as accurate as possible and test errors are minimized.
3.2 Description of test management
3.2.1 Preparation of the test field
Test plots are prepared according to the conventional operation of the crop under cultivation. Fields that can be harvested in time and are free of previous crop residues are carefully selected to ensure a high quality seedbed, because straw and roots remaining on the soil surface can easily entangle in the plow blade of the plot seeder, making it difficult to achieve precision in plot trials. Small-scale transgenic trials should not be conducted on plot test plots where conventional trials were previously done.
3.2.2 Fertilization
Fertilizers should be applied according to conventional tillage and production requirements. The amount of fertilizer should take into account the plant height of the transgenic plants. Since transgenic plants with high culms are prone to collapse, the amount of nitrogen fertilizer applied should be slightly less than the normal amount. Organic fertilizers should not be applied in small field trials to avoid uneven soil nutrient distribution.
3.2.3 Soil sterilization
This measure is limited to situations where there may be pest and weed hazards and should only be used if such treatments will not have an impact on the purpose of the trial.
3.2.4 Design and preparation of the experiment
The first step in conducting a trial is to develop a trial plan, identify the genotypes to be tested and their layout in the trial design, set the size of the trial area, number of replications, and trial protocol. The test seeds are prepared according to a sowing list, which includes details of the trial code, the code for each zone and the source of the seeds (which test zone of which trial). In addition, the variety name and pedigree of the genotype, or other identifying data are usually included. For some types of trials, the seeding list also indicates the number of rows sown and the number of seeds sown in each plot.
3.2.5 Seed preparation
How the seed for the test is prepared depends on factors such as how high or low the generation of the transgene is; also whether the genotype being tested was obtained by direct transformation or was bred from transgenic material; and how much seed is available. The spikes selected in the previous generation are threshed with a single-spike thresher, or manually, and the threshed seed is to be stored in a special container. All seeds are sorted in order according to the sowing list. If necessary, the seeds should also be cleaned with an experimental seed cleaner that avoids mixing of different genotypes. If necessary, the seeds can also be coated and divided into small bags according to the number of seeds sown in each plot. Before sowing, prepared seed should be stored separately from other test materials. When threshing, clearing, coating, and counting are complete, the experimental facility and room need to be thoroughly cleaned, and any excess seed and other plant tissue collected and destroyed.
3.2.6 Seeding
If only a small number of seeds are available, T1 and F1 generations, seeds can be sown by hand at wide spacing or with a single seed planter. For higher generations, such as T2, Tn or backcross-transferred segregating populations, single threshed seeds are sown individually into spike rows in the next generation. The Single Seeded Transmission (SSD) method is seldom used for field trials with additional generations. Transgenic pure populations are often sown into small plots using a specially designed trial plot seeder or spike row seeder. These machines are uniquely designed to sow all the seed loaded manually or automatically over a set distance. The density of sowing can be adjusted by varying the number of seeds or the sowing distance. In this way, seeds sown continuously in the field are not mixed and the machine does not need to be cleaned after sowing. The seeder will mulch and press the soil, etc., after sowing, so that it is not necessary to plow the field once again after sowing.
3.2.7 Segregation of transgenic field trials
The transgenic plants should be surrounded by at least 2 m wide conventional wheat varieties with at least 20 m wide open farmland or non-grain crop areas. These 2 m wide protected rows of wheat plants can be destroyed after the latest material in the trial has flowered and there is no longer a risk of crossbreeding between the transgenic wheat and the conventional wheat.
3.2.8 Disease-transmissible side rows
If the purpose of the field trial is to characterize the transgenic line for resistance to a fungus, the side rows must be planted with material consisting of susceptible genotypes in order to facilitate the induction of artificial inoculum. In tests to identify small species-specific diseases or insect pests, the side rows are to be planted with a mixture of genotypes. Direct inoculation of transgenic plants with laboratory-cultured strains is another method of identifying resistance [ 3 ], which involves inoculating a portion of the transgenic plants or a row of each plot.
3.2.9 Weed control and pest management
When the crop has emerged, the seedlings on the borders of the ends of the plots are trimmed to create the final plot size. This operation can be done by hand, trimmed with a rototiller, or sprayed with a broad-spectrum herbicide. Each plot is labeled with a plastic tag, which is printed with a thermal printer and secured to a sign post. The label needs to contain identifying information about the plot or other relevant information, and the label content should be both legible to the naked eye and recognizable by the barcode machine. When many plots are harvested at the same time, the plot labels can be removed and pasted onto the paper or cloth bags in which the seed is packaged to ensure that the plot numbers are not mistaken throughout the trial. If the climate of the trial site is suitable, it is recommended that a pre-emergence herbicide be sprayed immediately after sowing, especially important for thinly sown plots where they are highly susceptible to grass infestation. For grass and broadleaf weeds, common weed control measures are used and due attention is paid to the dominant grasses in the local weed community. Fungicides and insecticides are allowed for pest and disease control if not specifically required by the trial. Prior to maturity, plots were covered with netting to prevent seed dispersal by birds.
3.2.10 Irrigation
Usually wheat does not require watering. The only exception is watering to ensure that the transgenic plants survive drought conditions. Watering of test plots may also be required to promote disease development when testing transgenic plants for disease resistance. For some artificial inoculation tests, such as inoculation with wheat blast (FManum ), watering can also be used to ensure a high humidity environment in the field. Watering versus non-watering trials are also needed to test the effects of drought and heat adversity.
3.2.11 Examination and documentation
A field record book is organized according to the experimental design and is used to record the data collected in the field. The logbook should include all observations and measures from previous related trials, in addition to information such as the sample number for the current trial. In accordance with the trial protocol, each trial site was visited periodically throughout the growing season to record phenotypic data and compare them with the standard values of the non-transgenic. The phenotypic response of transgenic wheat to resistance to biological adversity should be observed and infection should be documented as soon as it is observed.
3.2.12 Harvesting
Depending on the needs of the experiment, either a plot test harvester is selected to harvest the entire plot or a single spike is selected for the transgenic lines. Harvested seeds are placed in paper or cloth bags and labeled accordingly to ensure accurate seed and numbering. The labels used to identify the plots can also be used as seed labels at harvest. Paper or cloth bags containing seed should be placed in a box or container with a closed lid to prevent seed from being spilled on the road. Transgenic wheat should be stored separately and individually from non-transgenic material and clearly labeled one by one. The amount of propagation obtained and the amount of seed used should be accurately recorded. To prevent mechanical mix-ups, the harvester should be thoroughly cleaned before leaving the trial area to ensure that no seed or spikes remain on the harvesting tables, drums and screens. Natural germination of scattered seed in the soil may be a cause of mechanical mixing between transgenic and conventional wheat; therefore, scattering should be minimized during harvest and efforts should be made to ensure that spikes are not left in the field.
3.2.13 Post-harvest handling
Residual plant material left in the field must be removed or plowed into the soil. The timing of tilling and plowing is determined by observing the germination of seeds left behind. Seeds that are left behind and germinate are removed by spraying with the appropriate herbicide or by tilling. In the following year, no more crops can be grown on this land, and in the third year, non-grain crops can be grown or the land can be left idle.
3.2.14 Emergency measures
In the unlikely event of the least expected accidents during the trial, or accidents such as spreading of plants, all plant material must be removed or destroyed immediately, and any material left behind must be tilled into the soil. If necessary, the plot and an area around the plot of about 2 m must also be sprayed with the appropriate herbicide. At the same time, the area must be monitored for replanting.
3.3 Environmental risk assessment for crop field trials
Risk assessment is an important part of the management of GM crop field trials. Particular attention should be paid to the risk of gene transfer, firstly, through pollen into other crops, or from plants into bacteria in the soil; and secondly, to the effect of the target or marker gene on other organisms. Because wheat is a self-pollinating crop, the probability of gene transfer does not appear to be high. Similarly, there have been no reports so far of negative effects occurring as a result of products of imported sequences. However, there is still a need to assess the potential probability of deleterious effects occurring and to keep an eye on the surrounding environmental conditions.
3.3.1 Survival, dispersal and ability to spread
Wheat is an annual plant, so it can only continue to produce offspring through the production of seeds. Spring wheat is usually sown in March and harvested in July or August, while winter wheat, whose sowing period varies from region to region, is often sown from September to late November, and matures 10 to 15 days earlier than spring wheat. Early harvesting minimizes the spread of material from animals or birds eating the seeds or ears. However, mature wheat kernels can fall to the ground before or during harvest and may survive the winter to germinate and grow the following spring. Straw is burned after harvest, and any plant residue in the field needs to be tilled into the soil. The genetically encoded proteins and the new components resulting from their action are rapidly degraded by the metabolism of soil microorganisms.
After harvest, dropped seeds are exposed after the stubble has been cut, and these can be tilled into the soil by another tillage operation so that they cannot survive for long periods of time. The probability of survival of leftover seeds can be minimized by treatments such as spraying the field with herbicides and removing all any regrowth. It is recommended that environmental release test plots be sprayed with glyphosate herbicide at a concentration of 3 L/hm2 within 10 days of harvest and removal of plant residues. If necessary, the herbicide can be sprayed twice. The following year, the trial area was monitored regularly and if any germinating wheat was found, it had to be hand-pulled and burned. The temperature of the soil also affects the survivability of the kernels. For example, for spring wheat, winter frosts can dramatically reduce the survivability of leftover kernels.
3.3.2 Potential for gene transfer to other wheat plants via pollen
Self-pollination of wheat under natural conditions is more than 99%, so the probability of natural hybridization of transgenic strains with other wheat varieties is very small. Wheat pollen grains are relatively heavy, and under normal conditions when they are picked up by the wind, they do not survive for more than 5-10 m, and the survival time is only 1-3 min, so cross-pollination occurs only under extreme weather conditions, and within a distance of 10-20 m at most.
3.3.3 Possibility of gene transfer to other species through pollen
There are concerns that transformed crops may become uncontrollable weeds, and that transferred genes will also spread to similar wild species such as weeds, resulting in extremely persistent weeds, so-called "superweeds". Although most crops do not cross with closely related species to produce offspring in most growing regions, there have been suggestions that all GM crops should be banned because of the potential for the introduced genes to spread to weeds.
The probability of natural hybridization is also very small ( about 1%) if wheat flowers at the same time as related wild species such as goatgrass, iceplant, ryegrass, barley, and pisum sativum. To date, there have been no reports of any naturally occurring hybrid plants between wheat and wild species, and if there may be any such hybrid progeny, they are either sterile or have very low viability. Ensuring that there are no plants associated with cultivars within 50 m of the experimental area also minimizes the possibility of cross-pollination. In particular, cultivated rye, which can hybridize with wheat, must not be planted in the immediate vicinity of the test site.
3.3.4 Possibility of gene transfer to microorganisms or animals
The risk of horizontal transfer of genes from plants to soil bacteria or gut bacteria is very low under natural conditions. The risk of gene transfer to human health is minimal compared to the occurrence of natural mutations in genes in bacteria, such as the spontaneous development of resistance mutations to ampicillin. The essential gene encoding β-glucosidase (GUS), which is often used as a marker gene for genetic transformation, is also widely present in microorganisms. Plasmid vectors used for transformation experiments often carry the ampicillin resistance gene, which cannot be expressed in transgenic plants because it is regulated by a prokaryotic promoter and is also present in soil bacteria. In the unlikely event that the ampicillin resistance gene is transferred horizontally into soil bacteria, the transformed soil bacteria will have a selective advantage only in a soil environment supplemented with the appropriate antibiotic.
3.3.5 Effect of expression products of imported sequences
Attention must be paid to the risks that may result from the product of the target gene, but the greatest risk of gene transfer still comes from the product of selectively labeled genes. Selective marker genes such as herbicide or antibiotic resistance are not genes that are required for transgenic plants; therefore, the focus of current research is on the development of new techniques to remove selective marker genes from transgenic plants. Removal of selective marker genes can be achieved by conventional gene isolation methods [ 9 ], however, homologous recombination is preferred to remove marker genes. The multiauto- transformation (MAT) vector system was developed for this purpose, and it can obtain marker-free transgenic plants in one simple step [ 10 ]. However, no potentially deleterious products have been reported in transgenic crops as a result of the introduced sequences, either in wheat or in other organisms in the environment.
3.3.6 Stability of Phenotypes and Genotypes
Tissue culture and transformation tests may produce phenotypic effects unrelated to transgenic effects, and such phenotypic effects could theoretically have an impact on field trials. Therefore, all transgenic lines for field release trials should be selected from phenotypically similar plants to the control to avoid phenotypic effects.
Mutations can also be caused by exogenous gene instability, and when developmental abnormalities are detected in transgenic plants, breeders need to use backcrosses with their parents to select strains that are phenotypically similar to the transformed parent. Therefore, all lines selected for field trials should be grown for several generations to obtain genetically stable lines.
3.3.7 Harmful effects on non-target organisms
Wheat is not normally a pathogenic source for humans or other species, and the pathogenicity of transformed plants, or their effects on humans and other organisms, do not manifest themselves differently from those of their parents or control varieties. Consumption of wheat products by allergic populations may result in certain adverse reactions, such as anaphylactic reactions, coeliac disease due to gluten intolerance, and dermatitis herpetiformis, while respiratory sensitivities, such as baker's asthma, may be caused by inhalation of flour. Respiratory allergies such as baker's asthma may be caused by inhaling flour. Similarly, wheat flour may cause hay fever allergy. However, there is no evidence that these deleterious effects are exacerbated in GM wheat. In our experience, the various deleterious effects of transgenic wheat behave in the same way as their control or non-transgenic plants.